Transgenic Underdominance: Pushing Transgenes into Populations.

Frank Crisione, Ph.D., Institute for Bioscience and Biotechnology Research, University of Maryland College Park

Insect population replacement and suppression using transgenic strategies are being considered for insects of medical and agricultural importance.

Rapidly increasing the frequency of transgenes in target populations after transgene release using genetic drive systems remains a significant challenge. Driving transgenes through target populations is an actively investigated field with factors such as geographic location, stability, and economic impact playing major roles in developing strategies.

Drosophila melanogaster

In addition to the current genetic drive systems, such as MEDEA, meiotic drive, homing endonucleases, Medusa, and CRISPR/Cas9-based systems, underdominance has been recently demonstrated as a potential mechanism for rapidly increasing transgene frequencies in populations (Reeves et al. , 2014).

Underdominance. Heterozygotes are less fit than either possible homozygotes. 01 vs 00 or 11

Although not exactly a gene drive system (it is more of a ‘push’ system), underdominance occurs when the fitness of heterozygotes at a particular locus is lower than both possible homozygotes. Under these conditions an unstable equilibrium is established that can be perturbed to result in all of one allele or the other in a population. The idea of utilizing underdominance as a method of transgene drive has been subjected to much theoretical but no empirical consideration (see for example, Magori & Gould 2006, Altrock et al., 2010).

For the first time, Reeves et al. (2014) have created an underdominance system in Drosophila melanogaster using two transgenes, albeit with some help of the Gal4/UAS system.

The authors silenced a gene known to show underdominance and then complemented the silenced genes with an almost identical transgene resistant to their silencing strategy.

Specifically, the authors produced an underdominance system through RNAi silencing of the haploinsufficient gene Rpl14, one of many Minute loci in D. melanogaster. Silencing this gene resulted in underdevelopment and reduced fertility that was rescued by a complementary transgene that was RNAi-refractory. However, a single rescue-allele is insufficient to reverse all the adverse effects of the knockdown (haploinsufficient), leading to heterozygous individuals with a severe fitness penalty.

Individuals homozygous for the rescue transgene suffer minimal fitness costs and persist in the population – a classic case of underdominance. The homozygous individuals are favored in the population and over generations if the unstable equilibrium is appropriately perturbed. Perturbing the equilibrium in this case was achieved by increasing the frequency of the silencing-resistant transgene. The authors report conversion of multiple small populations to being homozygous for the silencing-resistant transgene in as few as four generations.

Modeling the fate of the a allele starting with different initial frequencies. photo credit: http://hawaiireedlab.com

The unstable equilibrium associated with underdominance provides a reversibly means by which transgenes can be pushed through a population. This equilibrium is quickly shifted when one of the allele frequencies surpasses a critical threshold value (usually by transgene release). When the frequency of the transgene allele is above the threshold compared to the wild type allele then the mutant allele will “push” through the population and become fixed. The converse is true for wild type alleles, meaning that underdominant systems are bi-directional. An attractive feature of underdominance systems as a tool for introducing transgenes into natural populations is their intrinsic reversibility. Allelic frequencies can be shifted in favor of the transgene for a given time and then returned to wild type should the necessity arise.

Similar to any other gene drive mechanisms, allele frequency, fitness cost, and system stability (keep effector genes linked to the ‘driving’ alleles) are great concerns with using underdominance.

The bi-directionality of this system makes it very appealing from an ecological standpoint. However, it could potentially be a “double-edged sword” if the wild type allele manages, say by migration, to surpass the equilibrium threshold. It that happens then the population will revert to wild type.

This report is the first example of an artificial underdominance system constructed from transgenes and relies very much on the Gal4/UAS system. While this is useful as a laboratory demonstration, other solutions for regulating the expression of the silencing-transgene described by Reeves et al. (2014) will need to be found. Conditional systems like the Tet-Off system might be useful in this case.

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